Anti-adam28 antibody for treating cancer

ABSTRACT

The present invention provides an antibody that specifically binds to human ADAM28, inhibits enzyme activity of human ADAM28, and has an activity to suppress metastasis of a cancer cell that expresses human ADAM28. The antibody of the present invention can be a human antibody.

TECHNICAL FIELD

The present invention relates to an anti-human ADAM28 antibody, andpharmaceutical use thereof.

BACKGROUND ART

ADAM proteins (a disintegrin and metalloproteinases) are multifunctionalproteins involved in the ectodomain shedding of transmembrane proteins,cell adhesion and infiltration (non-patent documents 1, 2). The humangenome contains 25 ADAMs including four pseudogenes and 21 kinds ofADAMs are composed of 13 kinds of proteolytic ADAMs that exhibitproteolytic activity and eight kinds of non-proteolytic ADAMs(non-patent documents 1, 3). Proteolytic ADAMs share themetalloproteinase domain of matrix metalloproteinases (MMPs), and atypical proteolytic ADAM protein comprises propeptide,metalloproteinase, disintegrin-like, cysteine-rich, epidermal growthfactor-like, transmembranes and cytoplasmic domains (non-patentdocuments 3-9). Many proteolytic ADAMs, including ADAMS, ADAMS, ADAM12,ADAM15, ADAM17, ADAM19 and ADAM28 are overexpressed in human cancers andare associated with tumor growth and progression (non-patent documents5, 9). The present inventors' previous studies have indicated thatADAM28 (also known as ADAM metallopeptidase domain 28), which has twoalternative isoforms, including a prototype membrane-anchored form(ADAM28m) and a short secreted form (ADAM28s) (non-patent documents 5,10, 11), is abundantly expressed in human non-small cell lung and breastcarcinomas (non-patent documents 12, 13). By in situ hybridization andimmunohistochemistry, the present inventors have demonstrated thatADAM28 is expressed predominantly in carcinoma cells contained incarcinoma tissues and that the mRNA expression levels of ADAM28 areassociated with the cellular proliferation of breast cancer (non-patentdocument 13) and with both cancer cell proliferation and infiltration innon-small cell lung cancer (non-patent document 12). In a parallelstudy, the present inventors showed that serum ADAM27 levels innon-small cell lung cancer patients substantially increase with theprogression of tumor, lymph node metastasis, and cancer recurrence(non-patent document 14). These data imply that ADAM28 is involved incell proliferation and metastasis particularly in human cancer. Thepresent inventors have demonstrated that ADAM28 contributes to cancercell proliferation through enhanced bioavailability of insulin-likegrowth factor-I (IGF-I) by selective digestion of IGF-binding protein-3(IGFBP-3) of IGF-I/IGFBP-3 complex (non-patent document 13), and toangiogenesis by digestion of connective tissue growth factor in breastcancer (non-patent document 15).

The phage display method is one of the display techniques that haverealized a in vitro high-speed selection by forming a one-to-onecorrespondence in the form of phage particles between a functionalpeptide or protein and a DNA encoding same. This phage display methodhas been applied to antibody selection, and many antibodies obtained bythis method have been developed as medicaments (non-patent document 16).

Furthermore, a method of obtaining a specific antibody by combining ahuman artificial antibody library and a phage display method has alsobeen established, and such methods have been practicalized by pluralcompanies, as evidenced by HuCAL (Human Combinatorial Antibody Library)of MorphoSys.

DOCUMENT LIST Non-Patent Documents

-   non-patent document 1: Mol Aspects Med. 2008; 29 (5): 258-289-   non-patent document 2: Semin Cell Dev Biol. 2009; 20 (2): 138-145-   non-patent document 3: Pathol Int. 2010; 60 (7): 477-496-   non-patent document 4: Genes Dev. 2003; 17 (1): 7-30-   non-patent document 5: Cancer Sci. 2007; 98 (5): 621-628-   non-patent document 6: Nat Rev Mol Cell Biol. 2005; 6 (1): 32-43.-   non-patent document 7: Kelley's Textbook of Rheumatology. 8th ed.    Philadelphia, Pa.: Elsevier Saunders; 2009: 115-134-   non-patent document 8: Curr Opin Cell Biol. 2003; 15 (5): 598-606-   non-patent document 9: Nat Rev Cancer. 2008; 8 (12): 929-941-   non-patent document 10: J Biol Chem. 1999; 274 (41): 29251-29259-   non-patent document 11: Curr Pharm Des. 2009; 15 (20): 2349-2358-   non-patent document 12: Int J Cancer. 2006; 118 (2): 263-273-   non-patent document 13: Cancer Res. 2006; 66 (20): 9913-9920-   non-patent document 14: Int J Cancer. 2010; 127 (8): 1844-1856-   non-patent document 15: Biochem Biophys Res Commun. 2010; 402 (4):    651-657-   non-patent document 16: Rothe, C. et al. J. Mol. Biol. 2008;    376:1182-1200

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide an anti-human ADAM28antibody useful for the prophylaxis or treatment of cancer, orinhibition of cancer metastasis.

Means of Solving the Problems

To solve the above-mentioned problem, the present inventors haveprepared plural anti-ADAM28 antibodies that bond to human ADAM28. As aresult, they have found that some prepared anti-human ADAM28 antibodiesinhibit enzyme activity of ADAM28 and show a superior suppressive effecton cancer cell proliferation and an inhibitory effect on cancermetastasis in in vivo model. Based on the above findings, they haveconducted further studies and completed the present invention.

Accordingly, the present invention relates to the following.

[1] An antibody specifically binding to human ADAM28, and having anactivity to inhibit enzyme activity of human ADAM28.[2] The antibody of [1] that binds to human ADAM28 at an epitopecomprising the amino acid sequence shown in SEQ ID NO: 21, 22 or 23.[3] The antibody of [1], comprising a light chain variable region and aheavy chain variable region, wherein(1) the light chain variable region comprises CDR1 comprising the aminoacid sequence shown in SEQ ID NO: 5, CDR2 comprising the amino acidsequence shown in SEQ ID NO: 6 and CDR3 comprising the amino acidsequence shown in SEQ ID NO: 7, and the heavy chain variable regioncomprises CDR1 comprising the amino acid sequence shown in SEQ ID NO: 8,CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9 and CDR3comprising the amino acid sequence shown in SEQ ID NO: 10;(2) the light chain variable region comprises CDR1 comprising the aminoacid sequence shown in SEQ ID NO: 5, CDR2 comprising the amino acidsequence shown in SEQ ID NO: 6 and CDR3 comprising the amino acidsequence shown in SEQ ID NO: 7, and the heavy chain variable regioncomprises CDR1 comprising the amino acid sequence shown in SEQ ID NO: 8,CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9 and CDR3comprising the amino acid sequence shown in SEQ ID NO: 10, except that 1to 3 amino acids are substituted, deleted, inserted, and/or added in atleast one amino acid sequence selected from the group consisting of theamino acid sequences shown in SEQ ID NOs: 5, 6 and 7, and/or1 to 3 amino acids are substituted, deleted, inserted, and/or added inat least one amino acid sequence selected from the group consisting ofthe amino acid sequences shown in SEQ ID NOs: 8, 9 and 10;(3) the light chain variable region comprises CDR1 comprising the aminoacid sequence shown in SEQ ID NO: 11, CDR2 comprising the amino acidsequence shown in SEQ ID NO: 12 and CDR3 comprising the amino acidsequence shown in SEQ ID NO: 13, and the heavy chain variable regioncomprises CDR1 comprising the amino acid sequence shown in SEQ ID NO:14, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 15 andCDR3 comprising the amino acid sequence shown in SEQ ID NO: 16; or(4) the light chain variable region comprises CDR1 comprising the aminoacid sequence shown in SEQ ID NO: 11, CDR2 comprising the amino acidsequence shown in SEQ ID NO: 12 and CDR3 comprising the amino acidsequence shown in SEQ ID NO: 13, and the heavy chain variable regioncomprises CDR1 comprising the amino acid sequence shown in SEQ ID NO:14, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 15 andCDR3 comprising the amino acid sequence shown in SEQ ID NO: 16, exceptthat 1 to 3 amino acids are substituted, deleted, inserted, and/or addedin at least one amino acid sequence selected from the group consistingof the amino acid sequences shown in SEQ ID NOs: 11, 12 and 13, and/or1 to 3 amino acids are substituted, deleted, inserted, and/or added inat least one amino acid sequence selected from the group consisting ofthe amino acid sequences shown in SEQ ID NOs: 14, 15 and 16;(5) the light chain variable region comprises CDR1 comprising the aminoacid sequence shown in SEQ ID NO: 24, CDR2 comprising the amino acidsequence shown in SEQ ID NO: 25 and CDR3 comprising the amino acidsequence shown in SEQ ID NO: 26, and the heavy chain variable regioncomprises CDR1 comprising the amino acid sequence shown in SEQ ID NO:27, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 28 andCDR3 comprising the amino acid sequence shown in SEQ ID NO: 29; or(6) the light chain variable region comprises CDR1 comprising the aminoacid sequence shown in SEQ ID NO: 24, CDR2 comprising the amino acidsequence shown in SEQ ID NO: 25 and CDR3 comprising the amino acidsequence shown in SEQ ID NO: 26, and the heavy chain variable regioncomprises CDR1 comprising the amino acid sequence shown in SEQ ID NO:27, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 28 andCDR3 comprising the amino acid sequence shown in SEQ ID NO: 29,except that 1 to 3 amino acids are substituted, deleted, inserted,and/or added in at least one amino acid sequence selected from the groupconsisting of the amino acid sequences shown in SEQ ID NOs: 24, 25 and26, and/or 1 to 3 amino acids are substituted, deleted, inserted, and/oradded in at least one amino acid sequence selected from the groupconsisting of the amino acid sequences shown in SEQ ID NOs: 27, 28 and29.[4] The antibody of [3], wherein(1′) the light chain variable region comprises the amino acid sequenceshown in SEQ ID NO: 17, and the heavy chain variable region comprisesthe amino acid sequence shown in SEQ ID NO: 18;(3′) the light chain variable region comprises the amino acid sequenceshown in SEQ ID NO: 19, and the heavy chain variable region comprisesthe amino acid sequence shown in SEQ ID NO: 20; or(5′) the light chain variable region comprises the amino acid sequenceshown in SEQ ID NO: 30, and the heavy chain variable region comprisesthe amino acid sequence shown in SEQ ID NO: 31.[5] A pharmaceutical composition comprising the antibody of any of[1]-[4].[6] An agent for the prophylaxis or treatment of cancer, comprising theantibody of any of [1]-[4].[7] A cancer metastasis inhibitor comprising the antibody of any of[1]-[4].[8] A method of preventing or treating cancer in a mammal, comprisingadministering an effective amount of the antibody of any of [1]-[4] tothe mammal.[9] The method of [8], wherein the mammal is human.[10] A method of inhibiting cancer metastasis in a mammal, comprisingadministering an effective amount of the antibody of any of [1]-[4] tothe mammal.[11] The method of [10], wherein the mammal is human.[12] The antibody of any of [1]-[4] for use in the prophylaxis ortreatment of cancer.[13] The antibody of any of [1]-[4] for use in the inhibition of cancermetastasis.[14] Use of the antibody of any of [1]-[4] for the production of anagent for the prophylaxis or treatment of cancer.[15] Use of the antibody of any of [1]-[4] for the production of acancer metastasis inhibitor.[16] A polynucleotide encoding the antibody of any of [1]-[4].[17] A vector comprising the polynucleotide of [16].[18] A transformant comprising the vector of [17].

Effect of the Invention

According to the present invention, an anti-human ADAM28 antibody usefulfor the prophylaxis or treatment of cancer, or inhibition of cancermetastasis is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows of an enzyme activity inhibitory effect of anti-humanADAM28 antibody.

FIG. 2 shows an in vitro cancer cell proliferation inhibitory effect ofanti-human ADAM28 antibody.

FIG. 3 shows an in vivo cancer cell proliferation inhibitory effect ofanti-human ADAM28 antibody.

FIG. 4 shows an in vivo cancer cell metastasis inhibitory effect ofanti-human ADAM28 antibody.

FIG. 5 shows an in vivo cancer cell metastasis inhibitory effect ofanti-human ADAM28 antibody.

DESCRIPTION OF EMBODIMENTS

The present invention provides an antibody having a specific bindingactivity to human ADAM28, and an activity to inhibit enzyme activity ofhuman ADAM28.

ADAM28 is a known protein, and the amino acid sequence thereof and thecDNA sequence thereof are also known. ADAM28 includes two kinds of aprototype membrane-anchored form (ADAM28m) and a short secretion form(ADAM28s), both of which are encompassed in ADAM28 in the presentinvention. A representative amino acid sequence of human ADAM28m isshown in SEQ ID NO: 2, a representative cDNA sequence of human ADAM28mis shown in SEQ ID NO: 1, a representative amino acid sequence of humanADAM28s is shown in SEQ ID NO: 4, and a representative cDNA sequence ofhuman ADAM28s is shown in SEQ ID NO: 3.

The antibody of the present invention has a specific binding activity tohuman ADAM28.

The “human ADAM28” means that the amino acid sequence or nucleotidesequence of ADAM28 has the same or substantially the same amino acidsequence or nucleotide sequence as the amino acid sequence or nucleotidesequence of ADAM28 naturally expressed in human. Being “substantiallythe same” means that the amino acid sequence or nucleotide sequence ofinterest has not less than 70% (preferably not less than 80%, morepreferably not less than 90%, more preferably not less than 95%, mostpreferably not less than 99%), identity with the amino acid sequence ornucleotide sequence of ADAM28 naturally expressed in human, and has thefunction of human ADAM28. Terms for biological species other than human,proteins other than ADAM28, gene and fragments thereof are alsointerpreted in the same manner.

The “specific binding” of an antibody to antigen X means that thebinding affinity of an antibody to antigen X in an antigen-antibodyreaction is higher than the binding affinity to a non-specific antigen(e.g., bovine serum albumin (BSA)).

The antibody of the present invention has an activity to inhibit theenzymatic activity of human ADAM28. The enzyme activity of human ADAM28specifically means an activity of human ADAM28 to cleave human IGFBP-3(Insulin-like Growth Factor Binding Protein-3). The activity of humanADAM28 to cleave human IGFBP-3 can be evaluated by, for example, thezymography analysis described in Cancer Res 2006; 66(20):9913-9920.

In the present specification, the “antibody” is used as one encompassinga full-length antibody and any antigen-binding fragment (i.e.,“antigen-binding portion”) thereof or a single chain thereof. The“antibody” refers to a glycoprotein containing at least two heavy chains(H) and two light chains (L), which are linked by a disulfide bond, oran antigen-binding portion thereof. Each heavy chain is constituted by aheavy chain variable region (to be abbreviated as V_(H) herein) and aheavy chain constant region. The heavy chain constant region isconstituted by 3 domains of C_(H)1, C_(H)2 and C_(H)3. Each light chainis constituted by a light chain variable region (to be abbreviated asV_(L) herein) and a light chain constant region. The light chainconstant region is constituted by a single domain C_(L). V_(H) and V_(L)regions are further subdivided into regions with higher variabilitycalled complementarity determining regions (CDRs), which contain morehighly conservative regions called framework regions (FRs) scatteredtherein. Each V_(H) and V_(L) is constituted by 3 CDRs and 4 FRs, whichare aligned in the following order, i.e., FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4 from the amino terminus to the carboxy terminus. The variableregions of said heavy chain and light chain contain binding domains thatinteract with an antigen. The constant region of an antibody can mediatethe binding of immunoglobulin to host tissues or factors, includingvarious cells (e.g., effector cells) of the immune system and the firstcomponent (C1q) of the conventional complement system.

In the present specification, the “antigen-binding portion” of anantibody is used to refer to one or more fragments of an antibodyretaining an ability to specifically bind to an antigen (e.g., humanADAM28). It has been clarified that the antigen binding function of anantibody is performed by a fragment of a full-length antibody. Examplesof the binding fragment included in the term “antigen binding portion”of an antibody include (i) Fab fragment, a monovalent fragmentconstituted by V_(L), V_(H), C_(L) and C_(H1) domains, (ii) F(ab′)₂fragment, a divalent fragment containing two Fab fragments linked bydisulfide bond in the hinge region, (iii) Fab′ fragment, an inherent Fabhaving a hinge region portion (see FUNDAMENTAL IMMUNOLOGY, Paul ed., 3.sup. rd ed. 1993), (iv) Fd fragment constituted by V_(H) and C_(H1)domains, (v) Fv fragment constituted by V_(L) and V_(H) domains in asingle arm of an antibody, (vi) dAb fragment constituted by V_(H) domain(Ward et al., (1989) Nature 341:544-546), (vii) isolated complementaritydetermining region (CDR) and (viii) nanobody which is a heavy chainvariable region containing single variable domain and two constantregions. While V_(L) and V_(H), which are the two domains of Fvfragment, are encoded by different genes, they can be linked by asynthetic linker to produce a single protein chain from them byrecombinant techniques, wherein, in this chain, V_(L) and V_(H) regionspair with each other to form a monovalent molecule (known as a singlechain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibody is also encompassed in the“antigen-binding portion” of an antibody. Such antibody fragments areobtained by those of ordinary skill in the art by known conventionaltechniques, and screened for usefulness in the same manner as withunmodified antibody.

The antibody of the present invention is preferably a monoclonalantibody. The “monoclonal antibody” refers to a preparation of anantibody molecule of a single molecule composition. The monoclonalantibody composition shows single binding-specificity and affinity for aparticular epitope.

The antibody of the present invention is preferably a human antibody orhumanized antibody. The “human antibody” refers to an antibody havingvariable regions derived from a human germline immunoglobulin sequencein both, the framework and CDR regions. Furthermore, when an antibodycontains a constant region, the constant region also derives from ahuman germline immunoglobulin sequence. In the present specification,the “human antibody” also encompasses even an embodiment including anamino acid residue not encoded by a human germline immunoglobulinsequence (e.g., mutation introduced by random or site-directedmutagenesis in vitro or somatic mutation in vivo). In the presentspecification, in addition, the term of the “humanized antibody” meansan antibody wherein a CDR sequence derived from the germline of ananimal species other than human, such as mouse, is fused on the humanframework sequence.

In the present specification, the human antibody encompasses a“reconstituted human antibody”. The reconstituted human antibody refersto a modified antibody wherein at least one CDR contained in the firsthuman donor antibody is used in the second human acceptor antibody,instead of CDR of the second human acceptor antibody. Preferably, all 6CDRs are substituted. More preferably, the whole antigen binding region(e.g., Fv, Fab or F(ab′)2) of the first human donor antibody is usedinstead of the corresponding region in the second human acceptorantibody. More preferably, the Fab region of the first human donorantibody is operably linked to an appropriate constant region of thesecond human acceptor antibody to form a full-length antibody.

The reconstituted human antibody can be produced by conventional generecombinant techniques disclosed in, for example, EP125023, WO96/02576,the above-mentioned document 16 and the like. To be specific, forexample, a DNA sequence designed to link a desired CDR in a donor humanantibody and a desired framework region (FR) in an acceptor humanantibody is synthesized by PCR method using, as primers, severaloligonucleotides produced to have a region overlapping with the terminusregions of both CDR and FR (see the method described in WO98/13388). Theobtained. DNA is linked to a DNA encoding a human antibody constantregion or a human antibody constant region mutant, which is incorporatedinto a expression vector and the vector is introduced into a host toallow for production, whereby a reconstituted human antibody can beobtained (see EP125023, WO96/02576).

In the present specification, moreover, the human antibody encompassesan “artificial human antibody”. The artificial human antibody can beproduced by conventional gene recombinant techniques disclosed in, forexample, the above-mentioned document 16 and the like.

The antibody of the present invention also includes a fusion proteinwherein the aforementioned antibody and other peptide or protein arefused. The production method of a fusion protein includes linking apolynucleotide encoding the antibody of the present invention and apolynucleotide encoding other peptide or polypeptide to match the frame,introducing same into an expression vector, and allowing expressionthereof in a host, and techniques known to those of ordinary skill inthe art can be used. As other peptide to be fused with the antibody ofthe present invention, known peptides such as FLAG (Hopp, T. P. et al.,BioTechnology (1988) 6, 1204-1210), 6×His consisting of six His(histidine) residues, 10×His, human c-myc fragment, VSV-GP fragment,p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lcktag, α-tubulin fragment, B-tag, Protein C fragment and the like can beused. Examples of other polypeptide to be fused with the antibody of thepresent invention include GST (glutathione-S-transferase), HA (influenzahemagglutinin), immunoglobulin constant region, galactosidase, MBP(maltose binding protein) and the like. A commercially availablepolynucleotide encoding such peptide or polypeptide is fused with apolynucleotide encoding the antibody of the present invention, and afusion polynucleotide prepared thereby is expressed, whereby a fusionpolypeptide can be prepared.

The antibody of the present invention may be a conjugate antibody boundwith various molecules, for example, polymer substances such aspolyethylene glycol (PEG), hyaluronic acid and the like, radioactivesubstance, fluorescent substance, luminescence substance, enzyme, toxinand the like. Such conjugate antibody can be obtained by chemicallymodifying the obtained antibody. The modification method of antibody hasalready been established in this field (e.g., U.S. Pat. Nos. 5,057,313,5,156,840).

The antibody of the present invention is preferably isolated orpurified. Being “isolated or purified” means that an operation to removecomponents other than the component of interest has been applied to thestate of natural presence. The purity of the isolated or purifiedantibody of the present invention (ratio of the weight of the antibodyof the present invention to the total protein weight) is generally 50%or more, preferably 70% or more, more preferably 90% or more, mostpreferably 95% or more (e.g., substantially 100%).

In a preferable embodiment, the antibody of the present invention bindsto human ADAM28 in an epitope comprising the amino acid sequence shownin SEQ ID NO: 21, 22 or 23.

Examples of the epitope comprising the amino acid sequence shown in SEQID NO: 21 (ENFSKWRGS: hADAM28s 274-282) includes an epitope consistingof a continuous partial sequence of the amino acid sequence shown in SEQID NO: 4, which comprises the amino acid sequence shown in SEQ ID NO:21, and preferably has an amino acid length of 20 or less, morepreferably 12 or less. As the epitope comprising the amino acid sequenceshown in SEQ ID NO: 21, specifically, an epitope consisting of the aminoacid sequence shown in SEQ ID NO: 21, an epitope consisting of the aminoacid sequence shown in SEQ ID NO: 32 (FTLENFSKWRGS), and an epitopeconsisting of the amino acid sequence shown in SEQ ID NO: 33(ENFSKWRGSVLS) can be mentioned.

Examples of the epitope comprising the amino acid sequence shown in SEQID NO: 22 (TELWGPGRRT: hADAM28s 517-526) includes an epitope consistingof a continuous partial sequence of the amino acid sequence shown in SEQID NO: 4, which comprises the amino acid sequence shown in SEQ ID NO:22, and preferably has an amino acid length of 20 or less, morepreferably 12 or less. As the epitope comprising the amino acid sequenceshown in SEQ ID NO: 22, specifically, an epitope consisting of the aminoacid sequence shown in SEQ ID NO: 22 can be mentioned.

Examples of the epitope comprising the amino acid sequence shown in SEQID NO: 23 (LFNAPLPT: hADAM28s 395-402), includes an epitope consistingof a continuous partial sequence of the amino acid sequence shown in SEQID NO: 4, which comprises the amino acid sequence shown in SEQ ID NO:23, and preferably has an amino acid length of 20 or less, morepreferably 12 or less. As the epitope comprising the amino acid sequenceshown in SEQ ID NO: 23, specifically, an epitope consisting of the aminoacid sequence shown in SEQ ID NO: 23, an epitope consisting of the aminoacid sequence shown in SEQ ID NO: 34 (LSNCLFNAPLPT), and an epitopeconsisting of the amino acid sequence shown in SEQ ID NO: 35(CLFNAPLPTDII) can be mentioned.

As a preferable embodiment of the antibody of the present invention, theantibodies described in the following (1)-(4) can be mentioned:

(1) an antibody comprising a light chain variable region and a heavychain variable region,wherein the light chain variable region comprises CDR1comprising the amino acid sequence shown in SEQ ID NO: 5, CDR2comprising the amino acid sequence shown in SEQ ID NO: 6 and CDR3comprising the amino acid sequence shown in SEQ ID NO: 7, andthe heavy chain variable region comprises CDR1 comprising the amino acidsequence shown in SEQ ID NO: 8, CDR2 comprising the amino acid sequenceshown in SEQ ID NO: 9 and CDR3 comprising the amino acid sequence shownin SEQ ID NO: 10;(2) an antibody comprising a light chain variable region and a heavychain variable region,wherein the light chain variable region comprises CDR1 comprising theamino acid sequence shown in SEQ ID NO: 5, CDR2 comprising the aminoacid sequence shown in SEQ ID NO: 6 and CDR3 comprising the amino acidsequence shown in SEQ ID NO: 7, andthe heavy chain variable region comprises CDR1 comprising the amino acidsequence shown in SEQ ID NO: 8, CDR2 comprising the amino acid sequenceshown in SEQ ID NO: 9 and CDR3 comprising the amino acid sequence shownin SEQ ID NO: 10except that 1 to 3 amino acids are substituted, deleted, inserted,and/or added in at least one amino acid sequence selected from the groupconsisting of the amino acid sequences shown in SEQ ID NOs: 5, 6 and 7,and/or1 to 3 amino acids are substituted, deleted, inserted, and/or added inat least one amino acid sequence selected from the group consisting ofthe amino acid sequences shown in SEQ ID NOs: 8, 9 and 10.(3) an antibody comprising a light chain variable region and a heavychain variable region,wherein the light chain variable region comprises CDR1 comprising theamino acid sequence shown in SEQ ID NO: 11, CDR2 comprising the aminoacid sequence shown in SEQ ID NO: 12 and CDR3 comprising the amino acidsequence shown in SEQ ID NO: 13, andthe heavy chain variable region comprises CDR1 comprising the amino acidsequence shown in SEQ ID NO: 14, CDR2 comprising the amino acid sequenceshown in SEQ ID NO: 15 and CDR3 comprising the amino acid sequence shownin SEQ ID NO: 16;(4) an antibody comprising a light chain variable region and a heavychain variable region,wherein the light chain variable region comprises CDR1 comprising theamino acid sequence shown in SEQ ID NO: 11, CDR2 comprising the aminoacid sequence shown in SEQ ID NO: 12 and CDR3 comprising the amino acidsequence shown in SEQ ID NO: 13, andthe heavy chain variable region comprises CDR1 comprising the amino acidsequence shown in SEQ ID NO: 14, CDR2 comprising the amino acid sequenceshown in SEQ ID NO: 15 and CDR3 comprising the amino acid sequence shownin SEQ ID NO: 16,except that 1 to 3 amino acids are substituted, deleted, inserted,and/or added in at least one amino acid sequence selected from the groupconsisting of the amino acid sequences shown in SEQ ID NOs: 11, 12 and13, and/or1 to 3 amino acids are substituted, deleted, inserted, and/or added inat least one amino acid sequence selected from the group consisting ofthe amino acid sequences shown in SEQ ID NOs: 14, 15 and 16;(5) an antibody comprising a light chain variable region and a heavychain variable region,wherein the light chain variable region comprises CDR1 comprising theamino acid sequence shown in SEQ ID NO: 24, CDR2 comprising the aminoacid sequence shown in SEQ ID NO: 25 and CDR3 comprising the amino acidsequence shown in SEQ ID NO: 26, andthe heavy chain variable region comprises CDR1 comprising the amino acidsequence shown in SEQ ID NO: 27, CDR2 comprising the amino acid sequenceshown in SEQ ID NO: 28 and CDR3 comprising the amino acid sequence shownin SEQ ID NO 29; and(6) an antibody comprising a light chain variable region and a heavychain variable region,wherein the light chain variable region comprises CDR1 comprising theamino acid sequence shown in SEQ ID NO: 24, CDR2 comprising the aminoacid sequence shown in SEQ ID NO: 25 and CDR3 comprising the amino acidsequence shown in SEQ ID NO: 26, andthe heavy chain variable region comprises COR1 comprising the amino acidsequence shown in SEQ ID NO: 27, CDR2 comprising the amino acid sequenceshown in SEQ ID NO: 28 and CDR3 comprising the amino acid sequence shownin SEQ ID NO: 29,except that 1 to 3 amino acids are substituted, deleted, inserted,and/or added in at least one amino acid sequence selected from the groupconsisting of the amino acid sequences shown in SEQ ID NOs: 24, 25 and26, and/or1 to 3 amino acids are substituted, deleted, inserted, and/or added inat least one amino acid sequence selected from the group consisting ofthe amino acid sequences shown in SEQ ID NOs: 27, 28 and 29.

In the embodiments of (2), (4) and (6), the number of amino acids to besubstituted, deleted, inserted and/or added is not particularly limitedas long as the antibody has specific binding activity to human ADAM28,and has an activity to inhibit the enzyme activity of human ADAM28. Itis preferably within 2 amino acids, more preferably one amino acid, perone CDR sequence. While the number of CDR sequences in which amino acidis substituted, deleted, inserted and/or added is not particularlylimited as long as the antibody has specific binding activity to humanADAM28, and has an activity to inhibit the enzyme activity of ADAM28. Itis preferably within 2, more preferably one, per one light chainvariable region, and preferably within 2, more preferably 1, per oneheavy chain variable region. The substitution, deletion, insertionand/or addition of amino acid may be performed in both the light chainvariable region and the heavy chain variable region, or either one ofthem.

In the embodiments of (2), (4) and (6), 1-3 (preferably 1 or 2, morepreferably 1) amino acids are preferably substituted, deleted, inserted,and/or added only in the amino acid sequence of CDR3 in the light chainvariable region.

Examples of the method for substituting one or plural amino acidresidues with other desired amino acid include site-directed mutagenesismethod (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M.(1995) An oligodeoxyribonucleotide-directed dual amber method forsite-directed mutagenesis. Gene 152, 271-275; Zoller, M J, and Smith, M.(1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned intoM13 vectors. Methods Enzymol. 100, 468-500; Kramer, W, Drutsa, V,Jansen, H W, Kramer, B, Pflugfelder, M, and Fritz, H J (1984) The gappedduplex DNA approach to oligonucleotide-directed mutation construction.Nucleic Acids Res. 12, 9441-9456; Kramer W, and Fritz H J (1987)Oligonucleotide-directed construction of mutations via gapped duplex DNAMethods. Enzymol. 154, 350-367, Kunkel, T A (1985) Rapid and efficientsite-specific mutagenesis without phenotypic selection. Proc Natl AcadSci USA. 82, 488-492). Using these methods, desired amino acid in anantibody can be substituted by other amino acid of interest. Also, usingthe library technique such as framework shuffling (Mol Immunol. 2007April; 44(11):3049-60) and CDR repair (US2006/0122377) and the like, anamino acid in a framework or CDR can also be substituted by otherappropriate amino acid.

In the antibody of the present invention, as a framework region (FR) ofthe antibody to be linked to a CDR, a framework which enables the CDR toform a good antigen binding site is selected. While FR to be used forthe antibody of the present invention is not particularly limited andany FRs can be used, FR of a human antibody is preferably used. As theFR of a human antibody, one having a natural sequence may be used, orone or plural amino acids in the framework region having a naturalsequence may be substituted, deleted, added and/or inserted and the likeas necessary, so that CDR will form an appropriate antigen binding site.For example, a mutant FR sequence having desired properties can beselected by measuring and evaluating the binding activity of an antibodyhaving FR with substituted amino acid to an antigen (Sato, K. et al.,Cancer Res. (1993)53, 851-856).

In the antibodies of (1) and (2), FR of Vk1 (Kabat database) of humanantibody is preferably used for the light chain, and FR of VH5 (Kabatdatabase) of human antibody is preferably used for the heavy chain.

In the antibodies of (3) and (4), FR of Vk2 (Kabat database) of humanantibody is preferably used for the light chain, and FR of VH6 (Kabatdatabase) of human antibody is preferably used for the heavy chain.

In the antibodies of (5) and (6), FR of Vk2 (Kabat database) of humanantibody is preferably used for the light chain, and FR. of VH3 (Kabatdatabase) of human antibody is preferably used for the heavy chain.

The constant region used for the antibody of the present invention isnot particularly limited, and any constant region may be used.Preferable examples of the constant region used for the antibody of thepresent invention include constant regions of human antibody (constantregions derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM and the like). Forexample, Cγ1, Cγ2, Cy3, Cy4, Cμ, Cα1, Ca2, Cε can be used in H chain,and Cκ, Cλ can be used in L chain.

In the antibodies of (1)-(4), the constant region of CK of humanantibody is preferably used for the light chain, and the constant regionof Cγ1 of human antibody is preferably used for the heavy chain.

In the antibodies of (5) and (6), the constant region of Cκ of humanantibody is preferably used for the light chain, and the constant regionof Cγ1 of human antibody is preferably used for the heavy chain.

Preferable antibody of the present invention includes the following:

(1′) An antibody comprising a light chain variable region and a heavychain variable region, wherein the light chain variable region comprisesthe amino acid sequence shown in SEQ ID NO: 17 and the heavy chainvariable region comprises the amino acid sequence shown in SEQ ID NO:18;(3′) an antibody comprising a light chain variable region and a heavychain variable region, wherein the light chain variable region comprisesthe amino acid sequence shown in SEQ ID NO: 19 and the heavy chainvariable region comprises the amino acid sequence shown in SEQ ID NO:20; and(5′) an antibody comprising a light chain variable region and a heavychain variable region, wherein the light chain variable region comprisesthe amino acid sequence shown in SEQ ID NO: 30 and the heavy chainvariable region comprises the amino acid sequence shown in SEQ ID NO:31.

The antibody of the above-mentioned (1′) corresponds to a preferableembodiment of the antibody of the above-mentioned (1), and the antibodyof the above-mentioned (3′) corresponds to a preferable embodiment ofthe antibody of the above-mentioned (3), respectively. The antibody ofthe above-mentioned (5′) corresponds to a preferable embodiment of theantibody of the above-mentioned (5), respectively.

The present invention provides a polynucleotide containing a nucleotidesequence encoding the above-mentioned antibody of the present invention.The polynucleotide may be a DNA or RNA, or a DNA/RNA chimera. Thepolynucleotide may be double stranded or single stranded. When thepolynucleotide is double stranded, it may be a double stranded DNA, adouble stranded RNA or a DNA:RNA hybrid.

The polynucleotide of the present invention encompasses a polynucleotidecontaining a nucleotide sequence encoding both the heavy chain variableregion and the light chain variable region of the antibody of thepresent invention, and a combination of a polynucleotide containing anucleotide sequence encoding the heavy chain variable region of theantibody of the present invention and a polynucleotide containing anucleotide sequence encoding the light chain variable region of theantibody of the present invention.

The polynucleotide of the present invention can be easily produced basedon the information of the amino acid sequence of the antibody of thepresent invention, known sequence information and sequence informationdescribed in the Sequence Listing in the present specification, and byutilizing known gene recombination techniques. For example, suitableprimers are designed based on the sequence information, a DNA encodingthe elements constituting the antibody of the present invention isamplified by the PCR reaction, DNA fragments are ligated by appropriateenzymes such as ligase and the like, whereby the polynucleotide of thepresent invention can be produced. Alternatively, a polynucleotideencoding each element may be synthesized by a polynucleotidesynthesizer, based on the information of the amino acid sequence of theantibody of the present invention.

The obtained polynucleotide encoding the antibody of the presentinvention may be, depending on the object, directly used, or used afterdigestion with a restriction enzyme when desired, or addition of alinker. The polynucleotide may have ATG as a translation initiationcodon on the 5′ terminus side, and may have TAA, TGA or TAG as atranslation stop codon on the 3′ terminus side. These translationinitiation codon and translation stop codon can be added using asuitable synthesized DNA adapter.

The polynucleotide of the present invention is preferably isolated orpurified. The isolated or purified polynucleotide of the presentinvention has a purity (ratio of the weight of the polynucleotide of thepresent invention to the total polynucleotide weight) of generally 50%or more, preferably 70% or more, more preferably 90% or more, mostpreferably 95% or more (e.g., substantially 100%).

The present invention provides a vector comprising the above-mentionedpolynucleotide of the present invention. The vector of the presentinvention encompasses a vector comprising a polynucleotide comprising anucleotide sequence encoding both the heavy chain variable region andthe light chain variable region of the antibody of the presentinvention, and a combination of a vector comprising a polynucleotidecomprising a nucleotide sequence encoding the heavy chain variableregion of the antibody of the present invention and a vector comprisinga polynucleotide comprising a nucleotide sequence encoding the lightchain variable region of the antibody of the present invention. Thevector is preferably isolated or purified. Examples of the vectorinclude expression vector, cloning vector and the like, which can beselected according to the object. Preferably, the vector is anexpression vector. The expression vector can express the antibody of thepresent invention. The expression vector can be produced by operablylinking the polynucleotide of the present invention to the downstream ofa promoter in a suitable expression vector. The kind of the vectorincludes, for example, plasmid vector, virus vector and the like, whichcan be appropriately selected according to the host to be used.

As the host, the genus Escherichia (Escherichia coli etc.), the genusBacillus (Bacillus subtilis etc.), yeast (Saccharomyces cerevisiaeetc.), insect cell (established cell line derived from larva of Mamestrabrassicae (Spodoptera frugiperda cell; Sfcell) etc.), insect (larva ofBombyx mori etc.), mammalian cells (rat nerve cell, monkey cell (COS-7etc.), Chinese hamster cell (CHO cell etc.) etc.) and the like are used.

Examples of the mammal include, but are not limited to, experimentanimals such as rodents such as mouse, rat, hamster and guinea pig andthe like, rabbit and the like, domestic animals such as swine, bovine,goat, horse, sheep, mink and the like, companion animals such as dog,cat and the like, primates such as human, monkey, Macaca fascicularis,Macaca mulatta, marmoset, orangutan, chimpanzee and the like, and thelike.

Examples of the plasmid vector include plasmid vectors derived fromEscherichia coli (e.g., pBR322, pBR325, pUC12, pUC13), plasmid vectorsderived from Bacillus subtilis (e.g., pUB110, pTP5, pC194), plasmidvectors derived from yeast (e.g., pSH19, pSH15) and the like, which canbe appropriately selected according to the kind of the host to be usedand the object of use.

The kind of the virus vector can be appropriately selected according tothe kind of the host to be used and object of use. For example, when aninsect cell is used as a host, baculovirus vector and the like can beused. When a mammalian cell is used as a host, retrovirus vectors suchas moloney murine leukemia virus vector, lentivirus vector, sindbisvirus vector and the like, adenovirus vector, herpes virus vector,adeno-associated virus vector, parvovirus vector, vaccinia virus vector,sendai virus vector and the like can be used.

The promoter can be selected according to the kind of the host to beused, and one capable of initiating transcription in the host can beselected. For example, when the host is the genus Escherichia, trppromoter, lac promoter, T7 promoter and the like are preferable. Whenthe host is the genus Bacillus, SPO1 promoter, SPO2 promoter, penPpromoter and the like are preferable. When the host is yeast, PHO5promoter, PGK promoter and the like are preferable. When the host is aninsect cell, polyhedrin promoter, P10 promoter and the like arepreferable. When the host is a mammalian cell, subgenomic (26S)promoter, CMV promoter, SRα promoter and the like are preferable.

The vector of the present invention may contain a signal sequence forantibody secretion. As the signal sequence for antibody secretion whenit is produced in the periplasm of Escherichia coli, pelB signalsequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) may be used.

When desired, the vector of the present invention may contain enhancer,splicing signal, polyA addition signal, selection marker, SV40replication origin (hereinafter sometimes to be abbreviated as SV40ori)and the like each in an operable manner. Examples of the selectionmarker include dihydrofolate reductase (hereinafter sometimes to beabbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillinresistance gene (sometimes to be abbreviated as Amp^(r)), neomycinresistance gene (sometimes to be abbreviated as Neo^(r), G418resistance) and the like.

By introducing the above-mentioned vector of the present invention intothe above-mentioned host by gene transfer methods known per se (e.g.,lipofection method, calcium phosphate method, microinjection method,proplast fusion method, electroporation method, DEAE dextran method,gene transfer method by Gene Gun etc.), a transformant with the vectorintroduced thereinto (transformant of the present invention) can beproduced. When an expression vector is used as the vector to beintroduced, the transformant can express the antibody of the presentinvention. The transformant of the present invention is useful for theproduction of the antibody of the present invention and the like.

The antibody of the present invention can be produced by culturing thetransformant of the present invention by a method known per se accordingto the kind of the host, and isolating the antibody of the presentinvention from the culture. When the host is the genus Escherichia, thetransformant is cultured in an appropriate medium such as LB medium, M9medium and the like at generally about 15-43° C. for about 3-24 hr. Whenthe host is the genus Bacillus, the transformant is cultured in anappropriate medium generally at about 30-40° C. for about 6-24 hr. Whenthe host is yeast, the transformant is cultured in an appropriate mediumsuch as Burkholder's medium and the like generally at about 20° C.-35°C. for about 24-72 hr. When the host is an insect cell or insect, thetransformant is cultured in an appropriate medium such as Grace's Insectmedium added with about 10% of bovine serum and the like generally atabout 27° C. for about 3-5 days. When the host is an animal cell, thetransformant is cultured in an appropriate medium such as MEM mediumadded with about 10% of bovine serum and the like generally at about 30°C.-40° C. for about 15-60 hr. In any culture, aeration and stirring maybe performed as necessary.

As for the production method of antibody by genetic engineering, forexample, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M.and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A.and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E.,Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., MethodsEnzymol. (1986) 121, 663-669; Bird, R. E. and Walker, B. W., TrendsBiotechnol. (1991) 9, 132-137 and the like can be referred to.

The separation and purification of the antibody of the present inventionfrom a culture is not limited in any manner, and the separation andpurification methods generally used for purification of antibody can beemployed. For example, antibody can be separated and purified byappropriately selecting and combining chromatography column, filter,ultrafiltration, salting out, solvent precipitation, solvent extraction,distillation, immunoprecipitation, SDS-polyacrylamide gelelectrophoresis, isoelectric focusing, dialysis, recrystallization andthe like.

Examples of the chromatography include affinity chromatography, ionexchange chromatography, hydrophobic chromatography, gelfiltration,reversed-phase chromatography, adsorption chromatography and the like(Strategies for Protein Purification and Characterization: A LaboratoryCourse Manual. Ed Daniel R. Marshak et al., Cold Spring HarborLaboratory Press, 1996). These chromatographys can be performed by usingliquid phase chromatography, for example, liquid phase chromatographysuch as HPLC, FPLC and the like. Examples of the column to be used foraffinity chromatography include protein A column and protein G column.For example, as a column using protein A, Hyper D, POROS, Sepharose FF(manufactured by GE Amersham Biosciences) and the like can be mentioned.The present invention also encompasses an antibody highly purified bythese purification methods.

In addition, the present invention provides a pharmaceutical compositioncontaining the above-mentioned antibody of the present invention as anactive ingredient. The pharmaceutical composition of the presentinvention is useful as a prophylactic or therapeutic agent for cancer; acancer proliferation inhibitor; a cancer metastasis inhibitor and thelike. While the kind of cancer is not particularly limited as long as itcan achieve the prophylactic or therapeutic effect for cancer; cancerproliferation inhibitory effect; or cancer metastasis inhibitory effectby the antibody of the present invention, hepatic cancer, colorectalcancer, renal cancer, melanoma, pancreatic cancer, thyroid cancer,gastric cancer, lung cancer (small cell lung cancer, non-small cell lungcancer), brain tumor, uterine cancer, breast cancer, multipleosteosarcoma, ovarian cancer, chronic leukemia, prostate cancer, acutelymphoblastic leukemia, germinoma, acute myeloid leukemia, malignantlymphoma, villous cancer, pediatric malignant tumor, gall bladder orbile duct cancer and the like can be mentioned. In a preferableembodiment, the cancer to be the application target of the antibody ofthe present invention is a cancer that expresses human ADAM28. Whetherthe cancer expresses human ADAM28 can be evaluated by Western blotting,RT-PCR and the like.

While not bound by theories, ADAM28 inhibits formation of IGF-1/IGFBP-3complex by degrading IGFBP-3, and promotes cancer cell proliferation byIGF-1. The antibody of the present invention suppresses cancer cellproliferation by IGF-1 by inhibiting degradation of IGFBP-3 by ADAM28.In one embodiment, therefore, the cancer to be the application target ofthe antibody of the present invention is IGF-1 sensitive cancer (i.e.,cancer showing IGF-1-dependent proliferation promotion). Whether or notthe cancer is IGF-1 sensitive can be evaluated by analyzing theexpression of IGF-1 receptor in cancer by Western blotting, RT-PCR andthe like.

While not bound by theories, ADAM28 inhibits induction of apoptosis ofcancer cell by von Willebrand factor (vWF) by degrading vWF. Theantibody of the present invention promotes induction of apoptosis ofcancer cells by vWF by inhibiting degradation of vWF by ADAM28, as aresult of which it suppresses cancer proliferation and metastasis. Inone embodiment, therefore, the cancer to be the application target inthe present invention is vWF sensitive cancer (i.e., cancer permittinginduction of apoptosis by vWF). Whether or no the cancer is vWFsensitive can be evaluated by culturing the cancer cells on a vWF-coatedplate and analyzing the fragmentation of DNA by, for example, the methoddescribed in J Natl Cancer Inst 2012; 104:906-922.

When the antibody of the present invention is “contained as an activeingredient”, it means that the antibody of the present invention iscontained as at least one of the active ingredients, and does not limitthe content thereof. The pharmaceutical composition of the presentinvention may contain other active ingredient(s) together with theantibody of the present invention.

The antibody of the present invention can be formulated according to aconventional method (e.g., Remington's Pharmaceutical Science, latestedition, Mark Publishing Company, Easton, U.S.A.). Where necessary,moreover, it may contain a pharmaceutically acceptable carrier and/oradditive. For example, it can contain surfactant (PEG, Tween etc.),excipient, antioxidant (ascorbic acid etc.), colorant, flavor,preservative, stabilizer, buffering agent (phosphate, citrate, otherorganic acid etc.), chelating agent (EDTA etc.), suspending agent,isotonizing agent, binder, disintegrant, lubricant, glidant, corrigentand the like. Not being limited to these, the pharmaceutical compositionof the present invention may contain other conventional carriers asappropriate. Specific examples include light anhydrous silicic acid,lactose, crystalline cellulose, mannitol, starch, carmellose calcium,carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose,polyvinyl acetaldiethylaminoacetate, polyvinylpyrrolidone, gelatin,medium-chain fatty acid triglyceride, polyoxyethylene hydrogenatedcastor oil 60, sucrose, carboxymethylcellulose, cornstarch, inorganicsalts and the like. It may also contain other low-molecular-weightpolypeptide, serum albumin, gelatin and protein such as immunoglobulinand the like, as well as amino acid. When an aqueous solution forinjection is formulated, the antibody of the present invention isdissolved in, for example, isotonic solution containing saline, glucoseor other auxiliary agent. Examples of the auxiliary agent includeD-sorbitol, D-mannose, D-mannitol, and sodium chloride, and may be usedin combination with suitable solubilizing agents, for example, alcohol(ethanol etc.), polyalcohol (propylene glycol, PEG etc.), non-ionicsurfactant (polysorbate80, HCO-50) and the like.

Where necessary, polypeptide may also be included in a microcapsule(microcapsules made of hydroxymethylcellulose, gelatin,poly[methylmethacrylate] and the like), or formulated as a colloid drugdelivery system (liposome, albumin microsphere, microemulsion,nanoparticles and nanocapsule etc.) (see Remington's PharmaceuticalScience 16th edition &, Oslo Ed. (1980) etc.). Furthermore, a method offormulating a drug as a sustained-release medicament is also known, andapplicable to polypeptide (Langer et al., J. Biomed. Mater. Res.(1981)15: 167-277; Langer, Chem. Tech. (1982)12: 98-105; U.S. Pat. No.3,773,919; EP-A-58,481; Sidman et al., Biopolymers (1983) 22: 547-56; EPNo. 133,988). Furthermore, it is also possible to increase the liquidamount to be subcutaneously administered by adding or blendinghyaluronidase to or with the present agent (e.g., WO 2004/078140 etc.).

The content of the antibody of the present invention in a pharmaceuticalcomposition is, for example, about 0.01-100 wt %, preferably 0.1-99.9%,of the whole pharmaceutical composition.

While the pharmaceutical composition of the present invention can beadministered both orally and parenterally, it is preferably administeredparenterally. Specifically, it is administered to patients by injectionor transdermal administration. As an example of the dosage form ofinjection, it can be administered systemically or topically byintravenously injection, intramuscular injection, subcutaneous injectionand the like. It may also be administered to the treatment site or inthe vicinity thereof by topical injection, particularly intramuscularinjection. Examples of the dosage form of transdermal administrationinclude ointment, gel, cream, plaster, patch and the like, which can beadministered systemically or topically. In addition, the administrationmethod can be appropriately selected according to the age and symptom ofthe patients. The dose can be selected from, for example, the range of0.5 mg-10 mg/kg body weight as the antibody of the present invention.However, the pharmaceutical composition of the present invention is notlimited by these doses.

All references cited in the present specification, includingpublication, patent document and the like, are hereby incorporatedindividually and specifically by reference, to the extent that theentireties thereof have been specifically disclosed herein.

EXAMPLE

The present invention is explained in more detail in the following byreferring to Examples, which are not to be construed as limitative.Various gene manipulations in the Examples followed the method describedin Molecular cloning third. ed. (Cold Spring Harbor Lab. Press, 2001).

Example 1 Preparation of Antigen and Antibody

(1) Preparation of Human ADAM28 Recombinant Protein (rhADAM28)

Full length rhADAM28 was prepared and purified by the method describedin Biochem Biophys Res Commun. 2004; 315: 79-84.

(2) Biotinylation of rhADAM28

The purified rhADAM28 was biotinylated according to the standardprotocol of EZ-Link NHS-PEO₄-Biotin (Thermo Scientific), and theconcentration was determined by using BCA Protein Assay Kit(manufactured by PIERCE).

(3) Selection of Anti-Human ADMA28 Human Antibody Clones by PhageDisplay Method

The biotinylated rhADAM28 was immobilized on streptavidin-coatedmagnetic beads (Dynabeads MyOne Streptavidin T1 magnetic beads,manufactured by Invitrogen, 100 μl) at 4° C. for 1 hr, and washed 5times with 1 ml PBST (PBS containing 0.05% Tween 20). Using HuCAL GOLD(manufactured by MorphoSys) for human antibody phage library, antibodyselection was performed according to the method described in WO2007/042309, WO 2006/122797 and the like. rhADAM28-immobilized beadswere added to the phage library to bind an antigen-specific antibody.The magnetic beads were recovered and washed several times, and thephage was eluted from the magnetic beads. Escherichia coli cells wereinfected with the eluted phage and cultured at 37° C. overnight. Anoperation of phage-rescue from the phage-infected Escherichia coli cellsfollowed a general method (Molecular cloning third. Ed. Cold SpringHarbor Lab. Press, 2001). The selection round described above wasrepeated several times to concentrate a phage presenting an antibodyspecific to the antigen.

(4) Screening for anti-human ADMA28 human antibody by ELISA

The pool of Fab genes obtained after the concentration operation wassubcloned to Escherichia coli expression vector. According to the methoddescribed in WO 2006/122797 and the like, the Fab antibody wasexpressed, and the antigen-specific antibody was screened for by theELISA method. The Fab antibody was purified from a soluble fraction ofEscherichia coli lysate according to the standard method of Strep-Tactincolumn (manufactured by IBA). In addition, the purity of the purifiedantibody was confirmed by SDS-PAGE, and the concentration was determinedby using BCA Protein Assay Kit (manufactured by PIERCE).

(5) Analysis of Base Sequence of Anti-Human ADAM28 Human Antibody Clones

The obtained 2 clones (211-12, 211-14) of Escherichia coli werecultured, and plasmids were recovered (QIAprep Spin MiniPrep kit:manufactured by QIAGEN) and used for the base sequence analysis. Table 1shows the amino acid sequences of OCRs (complementarity determiningregions) of the respective clones. The full-length amino acid sequencesof the variable region of each clone are shown in SEQ ID NOs: 17-20.

TABLE 1 light chain LCDR1 LCDR2 LCDR3 211-12 RASQDI YGVS LQYD SSYLN TLQSSLPS (SEQ ID (SEQ ID (SEQ ID NO: 5) NO: 6) NO: 7) 211-14 RSSQSLLY YLGSFQYG SNGYIYLN NRAS GSPL (SEQ ID (SEQ ID (SEQ ID NO: 11) NO: 12) NO: 13)heavy chain HCDR1 HCDR2 HCDR3 211-12 YSFTS IIYPSDSYT WSWMG YWIA RYSPSFQGRGFDN (SEQ ID (SEQ ID (SEQ ID NO: 8) NO: 9) NO: 10) 211-14 DSVSSNVIYYRSKWY YKESIP TAAWG NDYAVSVKS EYGFDF (SEQ ID (SEQ ID (SEQ ID NO: 14)NO: 15) NO: 16)

(6) Preparation of IgG Antibody of Anti-Human ADAM28 Human AntibodyClones

Fab antibody genes of the obtained 2 clones were subcloned to constructIgG expression vectors (constant region of heavy chain was IgG1).HEK293T cells were transfected with these expression vectors accordingto the standard method of Lipofectamine (manufactured by Invitrogen),and the culture supernatant after culture for 72 hr was recovered. Asthe medium, DMEM (Sigma) supplemented with 10% Ultra Low IgG FBS(manufactured by Invitrogen) was used. From the culture supernatant, IgGantibody was purified by the standard method using rProteinA SepharoseFast Flow (manufactured by GE healthcare). Protein after purificationwas confirmed to show a single band by SDS-PAGE, and the concentrationwas determined by using BCA Protein Assay Kit (manufactured by PIERCE).

(7) Preparation of Anti-Human ADAM28 Mouse Monoclonal Antibody

Using purified rhADAM28 as an antigen, monoclonal antibodies to humanADAM28 protein were established. Five clones were first selected byELISA using rhADAM28, and clone 297-2F3 was selected as a candidateantibody to human ADAM28. The mono-reactivity of monoclonal antibody(297-2F3) was determined by immunoblotting for recombinant human ADAM28.

Example 2 Suppressive Effect of Anti-Human ADAM28 Antibodies on HumanADAM28 Enzyme Activity

rhADAM28 and anti-ADAM28 antibody were incubated at the weight ratioshown in the Figure for 2 hr, IGFBP-3 (100 ng) was added and incubatedat 37° C. for 24 hr, and the reaction was terminated with SDS-samplebuffer containing 5 mM EDTA. Thereafter, the reaction product wasapplied to SDS-PAGE (10% acrylamide gel), and the level of degradationof IGFBP-3 was detected by the immunoblot method using anti-IGFBP-3antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) (FIG. 1).Both 211-14 and 211-12 antibodies suppressed degradation of IGFBP-3 byhuman ADAM28.

Example 3

Proliferation suppressive effect of anti-human ADAM28 antibodies onbreast cancer cell line

The mitogenic effects of IGF-I on MDA-MB231 cells were measured with5-bromo-2′ deoxy-uridine (BrdUrd) Labeling and Detection Kit III (RocheMolecular Biochemicals, Basel, Switzerland) according to theinstructions of the manufacturer. After synchronization and growtharrest, cells were treated with 1 μg/mL IGF-I in DMEM containing 1% FBS.After synchronization and growth arrest, cells were treated with 1 μg/mLIGF-I in DMEM containing 1% FBS. After 6 hours, BrdUrd (10 μmol/L) wasadded to the media and cultured for the next 42 hours. To determine thecontribution of ADAM28 activity to the mitogenic effect of IGF-I, cellswere incubated with 1 or 5 μg/mL anti-ADAM28 antibody 30 minutes beforethe IGF-I treatment, and then reacted with BrdUrd for 42 hours in thepresence of IGF-I (FIG. 2). All of 211-14, 211-12 and 297-2F3 antibodiessuppressed in vitro growth of MDA-MB231 cells.

Example 4 Suppressive Effect of Anti-Human ADAM28 Antibodies on CancerCell Proliferation In Vivo

ADAM28 high expression breast cancer cell line MDA-MB231^(ffLuc-cp156)that constitutively expresses luciferase was prepared by a lentivirusvector, and transplanted (2×10e6 cells) to a breast subcutaneous tissueof NOD/SCID mouse (Six-week-old male, Charles River LaboratoriesInternational Inc, Washington, Mass.). After transplantation,anti-ADAM28 antibody (2 mg/kg/mice) was topically injected 5 times at 2day intervals, and the suppressive effect on tumor growth was examined.After intraperitoneal administration of D-luciferin (150 mg/Kg) (PromegaCo, Madison, Mich.), the luminescence was detected by in vivo imagingsystem (IVIS)-100 (Xenogen Co., Alameda, Calif.) (FIG. 3). Both 211-14and 211-12 antibodies suppressed in vivo proliferation ofMDA-MB231^(ffLuc-cp156).

Example 5 Suppressive Effect of Anti-Human ADAM28 Antibody on CancerCell Metastasis In Vivo

Male NOD/SCID mice (6 weeks old) (Charles River LaboratoriesInternational, Inc., Wilmington, Mass.) were injected withPC-9^(ffLuc-cp156) cells (1×10⁶ cells in 300 μl of PBS) into the tailvein. Lung metastasis was monitored by bioluminescence imaging using theIn Vivo Imaging System (IVIS)-100 camera system for detection ofluciferase activity (Xenogen Co., Alameda, Calif.) according to themanufacturer's instructions. During imaging, mice were anesthetized withisoflurane and received intraperitoneal injection of D-luciferin (150mg/kg; Promega Co., Madison, Wis.) and 1 minute later, photons from theanimal whole bodies were counted. To examine the effect of neutralizinganti-ADAM28 antibody on the lung metastasis, PC-9^(ffLuc-cp156) cellswere incubated with 5 μg/ml anti-human ADAM28 mouse monoclonal antibody(297-2F3) or 5 μg/ml non-immune IgG for 2 hours at 4° C. and thenintravenously injected into mice (n=9 mice per each group) (FIG. 4).Cancer metastasis was suppressed by 297-2F3 administration.

Example 6

In Example 4, RT-PCR with luciferase specific primers was performedusing RNA extracted from each organ of the mouse at 6 weeks aftertransplantation of cancer cells, the presence or absence oftumor-derived gene expression was confirmed, and the presence or absenceof inartificial metastasis was examined (FIG. 5). Both 211-14 and 211-12antibodies suppressed micrometastasis of cancer cells.

Example 7

With cDNA from hybridoma producing mouse anti-human ADAM28 monoclonalantibody 297-2F3, obtained in the above-mentioned Example 1(7), variableregions of the antibody were PCR-amplified, subcloned to a cloningvector, and the base sequence of the regions was analyzed. Table 2 showsthe amino acid sequences of the CDRs (complementary sex determinationregions). The full-length amino acid sequences of the light chain andheavy chain variable regions are respectively shown in SEQ ID NOs: 146and 147.

TABLE 2 light chain LCDR1 LCDR2 LCDR3 297- RSSQSLVL KISA SQTAH 2F3SNGNTYLN RFS VPWT (SEQ ID SEQ ID (SEQ ID NO: 24) (NO: 25) NO: 26)heavy chain HCDR1 HCDR2 HCDR3 297- DAWMD EIRSKANNHA FAY 2F3 (SEQ IDIYYAESVKG (SEQ ID NO: 27) (SEQ ID NO: 29) NO: 28)

Example 8

Then, by referring to the method described in WO 98/13388, mouseanti-human ADAM28 monoclonal antibody 297-2F3 was humanized by graftingeach CDR of the antibody to a human antibody frame. The full-lengthamino acid sequences of the light chain and heavy chain variable regionsof the thus-obtained humanized 297-2F3 are shown in SEQ ID NOs: 30 and31, respectively.

Example 9 Identification of Epitope (1)

Using a peptide array immobilized with partial peptides of humanADAM28s, epitope mapping was performed for the anti-human ADAM28antibodies 211-12 and 297-2F3. Specifically, as shown below, a peptidearray consisting of peptides having the residue number of 12 amino acidresidues with an offset of 3 amino acid residues were prepared for asequence covering from the protease domain to the C-terminal of humanADAM28s. HRP-labeled anti-human ADAM28s antibody was reacted with thepeptide array.

TABLE 3 1 VQEHEKYIEYYL (SEQ ID NO: 34) 2 HEKYIEYYLVLD (SEQ ID NO: 35) 3YIEYYLVLDNGE (SEQ ID NO: 36) 4 YYLVLDNGEFKR (SEQ ID NO: 37) 5VLDNGEFKRYNE (SEQ ID NO: 38) 6 NGEFKRYNENQD (SEQ ID NO: 39) 7FKRYNENQDEIR (SEQ ID NO: 40) 8 YNENQDEIRKRV (SEQ ID NO: 41) 9NQDEIRKRVFEM (SEQ ID NO: 42) 10 EIRKRVFEMANY (SEQ ID NO: 43) 11KRVFEMANYVNM (SEQ ID NO: 44) 12 FEMANYVNMLYE (SEQ ID NO: 45) 13ANYVNMLYKKIN (SEQ ID NO: 46) 14 VNMLYEKINTHV (SEQ ID NO: 47) 15INKELNTHVAIV (SEQ ID NO: 48) 16 KINTHVALVGME (SEQ ID NO: 49) 17THVALVGMEIWT (SEQ ID NO: 50) 18 ALVGMEIWTDKD (SEQ ID NO: 51) 19GMEIWTDKDKIK (SEQ ID NO: 52) 20 IWTDKDKIKITP (SEQ ID NO: 53) 21DKDKIKITPNAS (SEQ ID NO: 54) 22 KIKITPNASFTL (SEQ ID NO: 55) 23ITPNASFTLENF (SEQ ID NO: 56) 24 NASFTLENFSKW (SEQ ID NO: 57) 25FTLENFSKWRGS (SEQ ID NO: 58) 26 ENFSKWRGSVLS (SEQ ID NO: 59) 27SKWRGSVLSRRK (SEQ ID NO: 60) 28 RGSVISRRERHD (SEQ ID NO: 61) 29VLSRRKRHDIAQ (SEQ ID NO: 62) 30 RRKRHDIAQIIT (SEQ ID NO: 63) 31RHDIAQLITATE (SEQ ID NO: 64) 32 IAQLITATELAG (SEQ ID NO: 65) 33LITATELAGTTV (SEQ ID NO: 66) 34 ATELAGTTVGLA (SEQ ID NO: 67) 35LAGTTVGLAEMS (SEQ ID NO: 68) 36 TTVGLAFMSTMC (SEQ ID NO: 69) 37GLAFMSTMCSPY (SEQ ID NO: 70) 38 FMSTMCSPYSVG (SEQ ID NO: 71) 39TMCSPYSVGVVQ (SEQ ID NO: 72) 40 SPYSVGVVQDHS (SEQ ID NO: 73) 41SVGVVQDHSDNL (SEQ ID NO: 74) 42 VVQDHSDNLLRV (SEQ ID NO: 75) 43DHSDNLLRVAGT (SEQ ID NO: 76) 44 DNLLRVAGTMAH (SEQ ID NO: 77) 45LRVAGTMAHEMG (SEQ ID NO: 78) 46 AGTMAHEMGHNF (SEQ ID NO: 79) 47MAHEMGHNFGMF (SEQ ID NO: 80) 48 EMGHNFGMFHDD (SEQ ID NO: 81) 49HNFGMFHDDYSC (SEQ ID NO: 82) 50 GMFHDDYSCKCP (SEQ ID NO: 83) 51HDDYSCKCPSTI (SEQ ID NO: 84) 52 YSCKCPSTICVM (SEQ ID NO: 85) 53KCPSTICVMDKA (SEQ ID NO: 86) 54 STICVMDKALSF (SEQ ID NO: 87) 55CVMDKALSFYIP (SEQ ID NO: 88) 56 DKALSFYIPTDF (SEQ ID NO: 89) 57LSFYIPTDFSSC (SEQ ID NO: 90) 58 YIPTDFSSCSRI (SEQ ID NO: 91) 59TDESSCSRLSYD (SEQ ID NO: 92) 60 SSCSRLSYDKFF (SEQ ID NO: 93) 61SRLSYDKFFEDK (SEQ ID NO: 94) 62 SYDKFFEDKLSN (SEQ ID NO: 95) 63KEhEDKISNCLF (SEQ ID NO: 96) 64 EDKISNCLFNAP (SEQ ID NO: 97) 65LSNCLFNAPLPT (SEQ ID NO: 98) 66 CLFNAPLPTDII (SEQ ID NO: 99) 67NAPLPTDIISTP (SEQ ID NO: 100) 68 LPTDIISTPICG (SEQ ID NO: 101) 69DIISTPICGNQL (SEQ ID NO: 102) 70 STPICGNQLVEM (SEQ ID NO: 103) 71ICGNQLVEMGED (SEQ ID NO: 104) 72 NQLVEMGEDCDC (SEQ ID NO: 105) 73VEMGEDCDCGTS (SEQ ID NO: 106) 74 GEDCDCGTSEEC (SEQ ID NO: 107) 75CDCGTSEECTNI (SEQ ID NO: 108) 76 GTSEECTNICCD (SEQ ID NO: 109) 77EECTNICCDAKT (SEQ ID NO: 110) 78 TNICCDAKTCKI (SEQ ID NO: 111) 79CCDAKTCKIKAT (SEQ ID NO: 112) 80 AKTCKIKATFQC (SEQ ID NO: 113) 81CKIKATFQCALG (SEQ ID NO: 114) 82 KATFQCALGECC (SEQ ID NO: 115) 83FQCALGECCEKC (SEQ ID NO: 116) 84 ALGECCEKCQFK (SEQ ID NO: 117) 85ECCEKCQFKKAG (SEQ ID NO: 118) 86 EKCQFKKAGMVC (SEQ ID NO: 119) 87QFKKAGMVCRPA (SEQ ID NO: 120) 88 KAGMVCRPAKDE (SEQ ID NO: 121) 89MVCRPAKDECDL (SEQ ID NO: 122) 90 RPAKDECDLPEM (SEQ ID NO: 123) 91KDECDLPEMCNG (SEQ ID NO: 124) 92 CDLPEMCNGKSG (SEQ ID NO: 125) 93PEMCNGKSGNCP (SEQ ID NO: 126) 94 CNGKSGNCPDDR (SEQ ID NO: 127) 95KSGNCPDDRFQV (SEQ ID NO: 128) 96 NCPDDRFQVNGF (SEQ ID NO: 129) 97DDRFQVNGFPCH (SEQ ID NO: 130) 98 FQVNGFPCHHGK (SEQ ID NO: 131) 99NGFPCHHGKGHC (SEQ ID NO: 132) 100 PCHHGKGHCLMG (SEQ ID NO: 133) 101HGKGHCLMGTCP (SEQ ID NO: 134) 102 GHCLMGTCPTLQ (SEQ ID NO: 135) 103IMGTCPTLQEQC (SEQ ID NO: 136) 104 TCPTLQEQCTEL (SEQ ID NO: 137) 105TLQEQCTELWGP (SEQ ID NO: 138) 106 EQCTELWGPGRR (SEQ ID NO: 139) 107TELWGPGRRTNP (SEQ ID NO: 140) 108 WGPGRRTNPFPC (SEQ ID NO: 141) 109GRRTNPFPCACA (SEQ ID NO: 142) 110 TNPFPCACAKEN (SEQ ID NO: 143) 111FPCACAKENHFR (SEQ ID NO: 144)

As a result, 211-12 specifically bound to the above-mentioned peptides#25 and #26. The results suggest that the epitope of 211-12 contains theamino acid sequence shown in SEQ ID NO: 21 (ENFSKWRGS) common topeptides #25 and #26.

297-2F3 specifically bound to the above-mentioned peptides #65 and #66.In the consensus amino acid sequence (CLFNAPLPT: SEQ ID NO: 145) ofpeptides #65 and #66, cysteine is not recognized by antibodies in mostcases. Therefore, it is suggested that the epitope of 297-2F3 containsthe amino acid sequence shown in SEQ ID NO: 23 (LFNAPLPT). It wasconfirmed that 297-2F3 specifically recognizes SEQ ID NO: 23 as anepitope by dot blot using a lysate of Escherichia coli forciblyexpressing a fusion protein comprising the amino acid sequence shown inSEQ ID NO: 23 added to the C-terminal of MBP.

Example 9 Identification of Epitope (2)

Fusion proteins comprising a partial sequence of the following regionsof human ADAM28s (SEQ ID NO: 4) added to the C-terminal of MBP wererespectively prepared. 399-540, 399-497, 491-540, 491-510, 501-520,511-530, 521-540, 513-522, 515-524, 517-526, 519-528

As a control, a fusion protein comprising a partial sequence of thefollowing region of human ADAM28m (SEQ ID NO: 2) added to the C-terminalof MBP was prepared. 517-526 (human ADAM28m, as control)

The reactivity of anti-human ADAM28 antibody 211-14 to theabove-mentioned fusion proteins was evaluated by Western blotting. As aresult, 211-14 strongly bound to a fusion proteins containing a partialsequence of the regions 399-540, 491-540, 511-530 and 517-526 of humanADAM28s. The results suggest that the epitope of 211-14 contains the517-526 region (TELWGPGRRT, SEQ ID NO: 22) of human ADAM28s.

TABLE 4-1 human ADAM28m cDNA sequence SEQ ID NO: 1tcactggagaggaggcagggacagacccagcagcacccacctgagcgagaagagcagacaccgtgctcctggaatcacccagcatgttgcaaggtctcctgccagtcagtctcctcctctctgttgcagtaagtgctataaaagaactccctggggtgaagaagtatgaagtggtttatcctataagacttcatccactgcataaaagagaggccaaagagccagagcaacaggaacaatttgaaactgaattaaagtataaaatgacaattaatggaaaaattgcagtgctttatttgaaaaaaaacaagaacctccttgcaccaggctacacggaaacatattataattccactggaaaggagatcaccacaagcccacaaattatggatgattgttattatcaaggacatattcttaatgaaaaggtttctgacgctagcatcagcacatgtaggggtctaaggggctacttcagtcagggggatcaaagatactttattgaacctttaagccccatacatcgggatggacaggagcatgcactcttcaagtataaccctgatgaaaagaattatgacagcacctgtgggatggatggtgtgttgtgggcccacgatttgcagcagaacattgccctacctgccaccaaactagtaaaattgaaagacaggaaggttcaggaacatgagaaatacatagaatattatttggtcctggataatggtgagtttaaaaggtacaatgagaatcaagatgagatcagaaagagggtatttgagatggctaattatgtcaacatgctttataaaaagctcaatactcatgtggccttagttggtatggaaatctggactgacaaggataagataaagataaccccaaatgcaagcttcaccttggagaatttttctaaatggagggggagtgttctctcaagaagaaagcgtcatgatattgctcagttaatcacagcaacagaacttgctggaacgactgtgggtcttgcatttatgtctacaatgtgttctccttattctgttggcgttgttcaggaccacagcgataatcttcttagagttgcagggacaatggcacatgaaatgggccacaactttggaatgtttcatgacgactattcttgcaagtgtccttctacaatatgtgtgatggacaaagcactgagcttctatatacccacagacttcagttcctgcagccgtctcagctatgacaagttttttgaagataaattatcaaattgcctctttaatgctccattgcctacagatatcatatccactccaatttgtgggaaccagttggtggaaatgggagaggactgtgattgtgggacatctgaggaatgtaccaatatttgctgtgatgctaagacatgtaaaatcaaagcaacttttcaatgtgcattaggagaatgttgtgaaaaatgccaatttaaaaaggctgggatggtgtgcagaccagcaaaagatgagtgcgacctgcctgaaatgtgtaatggtaaatctggtaattgtcctgatgatagattccaagtcaatggcttcccttgccatcacgggaagggccactgcttgatggggacatgccccacactgcaggagcagtgcacagagctgtggggaccaggaactgaggttgcagataagtcatgttacaacaggaatgaaggtgggtcaaagtacgggtactgtcgcagagtggatgacacactcattccctgcaaagcaaatgataccatgtgtgggaagttgttctgtcaaggtgggtcggataatttgccctggaaaggacggatagtgactttcctgacatgtaaaacatttgatcctgaagacacaagtcaagaaataggcatggtggccaatggaactaagtgtggcgataacaaggtttgcattaatgcagaatgtgtggatattgagaaagcctacaaatcaaccaattgctcatctaagtgcaaaggacatgctgtgtgtgaccatgagctccagtgtcaatgtgaggaaggatggatccctcccgactgcgatgactcctcagtggtcttccacttctccattgtggttggggtgctgttcccaatggcggtcatttttgtggtggttgctatggtaatccggcaccagagctccagagaaaagcagaagaaagatcagaggccactatctaccactggcaccaggccacacaaacagaagaggaaaccccagatggtaaaggctgttcaaccccaagagatgagtcagatgaagccccatgtgtatgatctgccagtagaaggcaatgagcccccagcctcttttcataaagacacaaacgcacttccccctactgttttcaaggataatccagtgtctacacctaaggactcaaatccaaaagcatgaagcaacagctaagcaagaactaatggctaaattatcaacttggaaaactggaaaatctggatggcagagaaatatactatctatctcaccagtatttgctctcgactcaagaaggttaacattttctgattcatgttagactttgaagagactaaagaaaattttcaagaggaacatatgcctgagaacctttgcatgaatttaaaatttcaattatccattcttataagaaggaagatga

TABLE 4-2ttgtaaagaaatatctccgaagttaaaatctgtaataggaattgattcattctctaatgaaaacaaaacataaaaacatcacactaatcttggaggaataagaaaaattgtacatccattaaatgtacaattgattgcaacatcttgattgttttaaccattaacttgtcaaattacaatcacagttaagaaaatgatgtaaaattctgttttgtggatctctttcctagattagcttctgaaatcattattagctatatcatttgaggttttctacaatttggtataactaagaatttaaaaatgttttatcatatatatttgtataattaattactggcatggttaaagtggttttcactttttaaatggagaaaatttcagttaaattaataggataaaccaggttgcgaactggtgacctgtaggccatgtttgcactgcaaatatatttggtctgaatgatattgatattggacacatagtacttttacatgttttgaatgtattgctaatatttaaaaattgagagatcttgcataaacaatagattcccagctttgtcag a

TABLE 5 human ADAM28m amino acid sequence SEQ ID NO: 2MLQGLLPVSLLLSVAVSAIKELPGVKKYEVVYPIRLHPLHKREAKEPEQQEQFETELKYKMTINGKIAVLYLKKNKNLLAPGYTETYYNSTGKEITTSPQIMDDCYYQGHILNEKVSDASISTCRGLRGYFSQGDQRYFIEPLSPIHRDGQEHAISKYNPDEKNYDSTCGMDGVLWAHDLQQNIAIPATKLVKLKDRKVQEHEKYIEYYLVLDNGEFKRYNENQDEIRKRVFEMANYVNMLYKKLNTHVALVGMEIWTDKDKIKITPNASFTLENFSKWRGSVLSRRKRHDIAQLITATELAGTTVGLAFMSTMCSPYSVGVVQDHSDNLLRVAGTMAHEMGHNFGMFHDDYSCKCPSTICVMDKALSFYIPTDESSCSRLSYDKFFEDKLSNCLFNAPLPTDIISTPICGNQLVEMGEDCDCGTSEECTNICCDAKTCKIKATFQCALGECCEKCQFKKAGMVCRPAKDECDLPEMCNGKSGNCPDDRFQVNGFPCHHGKGHCLMGTCPTLQEQCTELWGPGTEVADKSCYNRNEGGSKYGYCRRVDDTLIPCKANDTMCGKLFCQGGSDNLPWKGRIVTFLTCKTFDPEDTSQEIGMVANGTKCGDNKVCINAECVDIEKAYKSTNCSSKCKGHAVCDHELQCQCEEGWIPPDCDDSSVVFHFSIVVGVLFPMAVIFVVVAMVIRHQSSREKQKKDQRPLSTTGTRPHKQKRKPQMVKAVQPQEMSQMKPHVYDLPVEGNEPPASFHKDTNALPPTVFKDNPVSTPKDSNPKA

TABLE 6 human ADAM28s cDNA sequence SEQ ID NO: 3tcactggagaggaggcagggacagacccagcagcacccacctgagcgagaagagcagacaccgtgctcctggaatcacccagcatgttgcaaggtctcctgccagtcagtetcctcctctctgttgcagtaagtgctataaaagaactccctggggtgaagaagtatgaagtggtttatcctataagacttcatccactgcataaaagagaggccaaagagccagagcaacaggaacaatttgaaactgaattaaagtataaaatgacaattaatggaaaaattgcagtgctttatttgaaaaaaaacaagaacctccttgcaccaggctacacggaaacatattataattccactggaaaggagatcaccacaagcccacaaattatggatgattgttattatcaaggacatattcttaatgaaaaggtttctgacgctagcatcagcacatgtaggggtctaaggggctacttcagtcagggggatcaaagatactttattgaacctttaagccccatacatcgggatggacaggagcatgcactcttcaagtataaccctgatgaaaagaattatgacagcacctgtgggatggatggtgtgttgtgggcccacgatttgcagcagaacattgccctacctgccaccaaactagtaaaattgaaagacaggaaggttcaggaacatgagaaatacatagaatattatttggtcctggataatggtgagtttaaaaggtacaatgagaatcaagatgagatcagaaagagggtatttgagatggctaattatgtcaacatgctttataaaaagctcaatactcatgtggccttagttggtatggaaatctggactgacaaggataagataaagataaccccaaatgcaagcttcaccttggagaatttttctaaatggagggggagtgttctctcaagaagaaagcgtcatgatattgctcagttaatcacagcaacagaacttgctggaacgactgtgggtcttgcatttatgtctacaatgtgttctccttattctgttggcgttgttcaggaccacagcgataatcttcttagagttgcagggacaatggcacatgaaatgggccacaactttggaatgtttcatgacgactattcttgcaagtgtccttctacaatatgtgtgatggacaaagcactgagcttctatatacccacagacttcagttcctgcagccgtctcagctatgacaagttttttgaagataaattatcaaattgcctctttaatgctccattgcctacagatatcatatccactccaatttgtgggaaccagttggtggaaatgggagaggactgtgattgtgggacatctgaggaatgtaccaatatttgctgtgatgctaagacatgtaaaatcaaagcaacttttcaatgtgcattaggagaatgttgtgaaaaatgccaatttaaaaaggctgggatggtgtgcagaccagcaaaagatgagtgcgacctgcctgaaatgtgtaatggtaaatctggtaattgtcctgatgatagattccaagtcaatggcttcccttgccatcacgggaagggccactgcttgatggggacatgccccacactgcaggagcagtgcacagagctgtggggaccaggtaggaggacaaatcctttcccctgtgcatgtgcgaaggaaaatcatttcagatgacagtgtttaaccatggtcaaaggaccattctgtcctatccttcttagaagctttgaactcaaaatcatggaaaggttttaagatttgaggttggttttagggttgctagatttagcaagtaaaaataaggatggccccgttaaattttaacttaaaattaacaagttttttgttaattttttgttttttgtctcagcatcagtatatcccatgcaatatttgaggtgtgctcatactaaaattatttgtgtatctgaaattcaaattaaactgggtgtotttttcttttcatctggcaaccctactaagatcataaacccttggaaatctgtgtgtgtgcgggtgtgtgtgtgtgtgtgtgtgcaggggtggcagaagtactgtgggatgggacagaaataa

TABLE 7 human ADAM28s amino acid sequence SEQ ID NO: 4MLQGLLPVSLLLSVAVSAIKELPGVKKYEVVYPIRLHPLHKREAKEPEQQEQFETELKYKMTINGKIAVLYLKKNKNLLAPGYTETYYNSTGKEITTSPQIMDDCYYQGHILNEKVSDASISTCRGLRGYFSQGDQRYFIEPLSPIHRDGQEHALFKYNPDEKNYDSTCGMDGVLWAHDLQQNIALPATKLVKLKDRKVQEHEKYIEYYLVLDNGEFKRYNENQDEIRKRVFEMANYVNMLYKKLNTHVALVGMEIWTDKDKIKITPNASFTLENFSKWRGSVLSRRKRHDIAQLITATELAGTTVGLAFMSTMCSPYSVGVVQDHSDNLLRVAGTMAHEMGHNFGMFHDDYSCKCPSTICVMDKALSFYIPTDFSSCSRLSYDKFFEDKLSNCLFNAPLPTDIISTPICGNQLVEMGEDCDCGTSEECTNICCDAKTCKIKATFQCALGECCEKCQFKKAGMVORPAKDECDLPEMONGKSGNOPDDRFQVNGFPCHHGKGHCLMGTCPTLQEQCTELWGPGRRTNPFPCACAKENHFR

TABLE 8 211-12 VL (kappal) SEQ ID NO: 17DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYGVSTLQSGVPSRESGSGSGTDFTLTISSLQPEDFATYYCLQYDSL PSTEGQGTKVEIKRT

TABLE 9 211-12 VH (VH5) SEQ ID NO: 18QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEWMGIIYPSDSYTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYC ARWSWMGRGEDNWGQGTLVTVSS

TABLE 10 211-14 VL (kappa2) SEQ ID NO: 19DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYTYLNWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQY GGSPLTFGQGTKVEIKRT

TABLE 11 211-12 VH (VH6) SEQ ID NO: 20QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNTAAWGWIRQSPGRGLEWLGVIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARYKESIPEYGFDFWGQGTLVTVSS

TABLE 12 humanized 297-2F3 VL (VLk2) SEQ ID NO: 30DIVMTQSPLSLPVTLGQPASISCRSSQSLVLSNGNTYLNWFQQRPGQSPRLLIYKISARFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQT AHVPWTFGGGTKVEIKRT

TABLE 13 humanized 297-2F3 VH (VH3) SEQ ID NO: 31QVQLVESGGGLVKPGGSLRLSCAASGFTFSDAWMDWVRQAPGKGLEWVGEIRSKANNHAIYYAESVKGRFTISRDDSENTLYLQMNSLKTEDTA VYYCTGFAYWGQGTLVTVSS

INDUSTRIAL APPLICABILITY

According to the present invention, an anti-human ADAM28 antibodyapplicable to the prophylaxis or treatment of cancer is provided.

This application is based on a provisional patent application No.61/724,484 filed in U.S.A. (filing date: Nov. 9, 2012), the contents ofwhich are incorporated in full herein.

1. An antibody specifically binding to human ADAM28, and having anactivity to inhibit enzyme activity of human ADAM28.
 2. The antibodyaccording to claim 1 that binds to human ADAM28 at an epitope comprisingthe amino acid sequence shown in SEQ ID NO: 21, 22 or
 23. 3. Theantibody according to claim 1, comprising a light chain variable regionand a heavy chain variable region, wherein (1) the light chain variableregion comprises CDR1 comprising the amino acid sequence shown in SEQ IDNO: 5, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 6 andCDR3 comprising the amino acid sequence shown in SEQ ID NO: 7, and theheavy chain variable region comprises CDR1 comprising the amino acidsequence shown in SEQ ID NO: 8, CDR2 comprising the amino acid sequenceshown in SEQ ID NO: 9 and CDR3 comprising the amino acid sequence shownin SEQ ID NO: 10; (2) the light chain variable region comprises CDR1comprising the amino acid sequence shown in SEQ ID NO: 5, CDR2comprising the amino acid sequence shown in SEQ ID NO: 6 and CDR3comprising the amino acid sequence shown in SEQ ID NO: 7, and the heavychain variable region comprises CDR1 comprising the amino acid sequenceshown in SEQ ID NO: 8, CDR2 comprising the amino acid sequence shown inSEQ ID NO: 9 and CDR3 comprising the amino acid sequence shown in SEQ IDNO: 10, except that 1 to 3 amino acids are substituted, deleted,inserted, and/or added in at least one amino acid sequence selected fromthe group consisting of the amino acid sequences shown in SEQ ID NOs: 5,6 and 7, and/or 1 to 3 amino acids are substituted, deleted, inserted,and/or added in at least one amino acid sequence selected from the groupconsisting of the amino acid sequences shown in SEQ ID NOs: 8, 9 and 10;(3) the light chain variable region comprises CDR1 comprising the aminoacid sequence shown in SEQ ID NO: 11, CDR2 comprising the amino acidsequence shown in SEQ ID NO: 12 and CDR3 comprising the amino acidsequence shown in SEQ ID NO: 13, and the heavy chain variable regioncomprises CDR1 comprising the amino acid sequence shown in SEQ ID NO:14, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 15 andCDR3 comprising the amino acid sequence shown in SEQ ID NO: 16; or (4)the light chain variable region comprises CDR1 comprising the amino acidsequence shown in SEQ ID NO: 11, CDR2 comprising the amino acid sequenceshown in SEQ ID NO: 12 and CDR3 comprising the amino acid sequence shownin SEQ ID NO: 13, and the heavy chain variable region comprises CDR1comprising the amino acid sequence shown in SEQ ID NO: 14, CDR2comprising the amino acid sequence shown in SEQ ID NO: 15 and CDR3comprising the amino acid sequence shown in SEQ ID NO: 16, except that 1to 3 amino acids are substituted, deleted, inserted, and/or added in atleast one amino acid sequence selected from the group consisting of theamino acid sequences shown in SEQ ID NOs: 11, 12 and 13, and/or 1 to 3amino acids are substituted, deleted, inserted, and/or added in at leastone amino acid sequence selected from the group consisting of the aminoacid sequences shown in SEQ ID NOs: 14, 15 and 16; (5) the light chainvariable region comprises CDR1 comprising the amino acid sequence shownin SEQ ID NO: 24, CDR2 comprising the amino acid sequence shown in SEQID NO: 25 and CDR3 comprising the amino acid sequence shown in SEQ IDNO: 26, and the heavy chain variable region comprises CDR1 comprisingthe amino acid sequence shown in SEQ ID NO: 27, CDR2 comprising theamino acid sequence shown in SEQ ID NO: 28 and CDR3 comprising the aminoacid sequence shown in SEQ ID NO: 29; or (6) the light chain variableregion comprises CDR1 comprising the amino acid sequence shown in SEQ IDNO: 24, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 25and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 26, andthe heavy chain variable region comprises CDR1 comprising the amino acidsequence shown in SEQ ID NO: 27, CDR2 comprising the amino acid sequenceshown in SEQ ID NO: 28 and CDR3 comprising the amino acid sequence shownin SEQ ID NO: 29, except that 1 to 3 amino acids are substituted,deleted, inserted, and/or added in at least one amino acid sequenceselected from the group consisting of the amino acid sequences shown inSEQ ID NOs: 24, 25 and 26, and/or 1 to 3 amino acids are substituted,deleted, inserted, and/or added in at least one amino acid sequenceselected from the group consisting of the amino acid sequences shown inSEQ ID NOs: 27, 28 and
 29. 4. The antibody according to claim 3, wherein(1′) the light chain variable region comprises the amino acid sequenceshown in SEQ ID NO: 17, and the heavy chain variable region comprisesthe amino acid sequence shown in SEQ ID NO: 18; (3′) the light chainvariable region comprises the amino acid sequence shown in SEQ ID NO:19, and the heavy chain variable region comprises the amino acidsequence shown in SEQ ID NO: 20; or (5′) the light chain variable regioncomprises the amino acid sequence shown in SEQ ID NO: 30, and the heavychain variable region comprises the amino acid sequence shown in SEQ IDNO:
 31. 5. A pharmaceutical composition comprising the antibodyaccording to claim
 1. 6.-7. (canceled)
 8. A method of preventing ortreating cancer in a mammal, comprising administering an effectiveamount of the antibody according to claim 1 to the mammal.
 9. The methodaccording to claim 8, wherein the mammal is human.
 10. A method ofinhibiting cancer metastasis in a mammal, comprising administering aneffective amount of the antibody according to claim 1 to the mammal. 11.The method according to claim 10, wherein the mammal is human. 12.-15.(canceled)
 16. A polynucleotide encoding the antibody according toclaim
 1. 17. A vector comprising the polynucleotide according to claim16.
 18. A transformant comprising the vector according to claim
 17. 19.The method according to claim 8, wherein the antibody binds to humanADAM28 at an epitope comprising the amino acid sequence shown in SEQ IDNO: 21, 22 or
 23. 20. The method according to claim 8, wherein theantibody comprises a light chain variable region and a heavy chainvariable region, wherein (1) the light chain variable region comprisesCDR1 comprising the amino acid sequence shown in SEQ ID NO: 5, CDR2comprising the amino acid sequence shown in SEQ ID NO: 6 and CDR3comprising the amino acid sequence shown in SEQ ID NO: 7, and the heavychain variable region comprises CDR1 comprising the amino acid sequenceshown in SEQ ID NO: 8, CDR2 comprising the amino acid sequence shown inSEQ ID NO: 9 and CDR3 comprising the amino acid sequence shown in SEQ IDNO: 10; (2) the light chain variable region comprises CDR1 comprisingthe amino acid sequence shown in SEQ ID NO: 5, CDR2 comprising the aminoacid sequence shown in SEQ ID NO: 6 and CDR3 comprising the amino acidsequence shown in SEQ ID NO: 7, and the heavy chain variable regioncomprises CDR1 comprising the amino acid sequence shown in SEQ ID NO: 8,CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9 and CDR3comprising the amino acid sequence shown in SEQ ID NO: 10, except that 1to 3 amino acids are substituted, deleted, inserted, and/or added in atleast one amino acid sequence selected from the group consisting of theamino acid sequences shown in SEQ ID NOs: 5, 6 and 7, and/or 1 to 3amino acids are substituted, deleted, inserted, and/or added in at leastone amino acid sequence selected from the group consisting of the aminoacid sequences shown in SEQ ID NOs: 8, 9 and 10; (3) the light chainvariable region comprises CDR1 comprising the amino acid sequence shownin SEQ ID NO: 11, CDR2 comprising the amino acid sequence shown in SEQID NO: 12 and CDR3 comprising the amino acid sequence shown in SEQ IDNO: 13, and the heavy chain variable region comprises CDR1 comprisingthe amino acid sequence shown in SEQ ID NO: 14, CDR2 comprising theamino acid sequence shown in SEQ ID NO: 15 and CDR3 comprising the aminoacid sequence shown in SEQ ID NO: 16; or (4) the light chain variableregion comprises CDR1 comprising the amino acid sequence shown in SEQ IDNO: 11, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 12and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13, andthe heavy chain variable region comprises CDR1 comprising the amino acidsequence shown in SEQ ID NO: 14, CDR2 comprising the amino acid sequenceshown in SEQ ID NO: 15 and CDR3 comprising the amino acid sequence shownin SEQ ID NO: 16, except that 1 to 3 amino acids are substituted,deleted, inserted, and/or added in at least one amino acid sequenceselected from the group consisting of the amino acid sequences shown inSEQ ID NOs: 11, 12 and 13, and/or 1 to 3 amino acids are substituted,deleted, inserted, and/or added in at least one amino acid sequenceselected from the group consisting of the amino acid sequences shown inSEQ ID NOs: 14, 15 and 16; (5) the light chain variable region comprisesCDR1 comprising the amino acid sequence shown in SEQ ID NO: 24, CDR2comprising the amino acid sequence shown in SEQ ID NO: 25 and CDR3comprising the amino acid sequence shown in SEQ ID NO: 26, and the heavychain variable region comprises CDR1 comprising the amino acid sequenceshown in SEQ ID NO: 27, CDR2 comprising the amino acid sequence shown inSEQ ID NO: 28 and CDR3 comprising the amino acid sequence shown in SEQID NO: 29; or (6) the light chain variable region comprises CDR1comprising the amino acid sequence shown in SEQ ID NO: 24, CDR2comprising the amino acid sequence shown in SEQ ID NO: 25 and CDR3comprising the amino acid sequence shown in SEQ ID NO: 26, and the heavychain variable region comprises CDR1 comprising the amino acid sequenceshown in SEQ ID NO: 27, CDR2 comprising the amino acid sequence shown inSEQ ID NO: 28 and CDR3 comprising the amino acid sequence shown in SEQID NO: 29, except that 1 to 3 amino acids are substituted, deleted,inserted, and/or added in at least one amino acid sequence selected fromthe group consisting of the amino acid sequences shown in SEQ ID NOs:24, 25 and 26, and/or 1 to 3 amino acids are substituted, deleted,inserted, and/or added in at least one amino acid sequence selected fromthe group consisting of the amino acid sequences shown in SEQ ID NOs:27, 28 and
 29. 21. The method according to claim 20, wherein (1′) thelight chain variable region comprises the amino acid sequence shown inSEQ ID NO: 17, and the heavy chain variable region comprises the aminoacid sequence shown in SEQ ID NO: 18; (3′) the light chain variableregion comprises the amino acid sequence shown in SEQ ID NO: 19, and theheavy chain variable region comprises the amino acid sequence shown inSEQ ID NO: 20; or (5′) the light chain variable region comprises theamino acid sequence shown in SEQ ID NO: 30, and the heavy chain variableregion comprises the amino acid sequence shown in SEQ ID NO:
 31. 22. Themethod according to claim 10, wherein the antibody binds to human ADAM28at an epitope comprising the amino acid sequence shown in SEQ ID NO: 21,22 or
 23. 23. The method according to claim 10, wherein the antibodycomprises a light chain variable region and a heavy chain variableregion, wherein (1) the light chain variable region comprises CDR1comprising the amino acid sequence shown in SEQ ID NO: 5, CDR2comprising the amino acid sequence shown in SEQ ID NO: 6 and CDR3comprising the amino acid sequence shown in SEQ ID NO: 7, and the heavychain variable region comprises CDR1 comprising the amino acid sequenceshown in SEQ ID NO: 8, CDR2 comprising the amino acid sequence shown inSEQ ID NO: 9 and CDR3 comprising the amino acid sequence shown in SEQ IDNO: 10; (2) the light chain variable region comprises CDR1 comprisingthe amino acid sequence shown in SEQ ID NO: 5, CDR2 comprising the aminoacid sequence shown in SEQ ID NO: 6 and CDR3 comprising the amino acidsequence shown in SEQ ID NO: 7, and the heavy chain variable regioncomprises CDR1 comprising the amino acid sequence shown in SEQ ID NO: 8,CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9 and CDR3comprising the amino acid sequence shown in SEQ ID NO: 10, except that 1to 3 amino acids are substituted, deleted, inserted, and/or added in atleast one amino acid sequence selected from the group consisting of theamino acid sequences shown in SEQ ID NOs: 5, 6 and 7, and/or 1 to 3amino acids are substituted, deleted, inserted, and/or added in at leastone amino acid sequence selected from the group consisting of the aminoacid sequences shown in SEQ ID NOs: 8, 9 and 10; (3) the light chainvariable region comprises CDR1 comprising the amino acid sequence shownin SEQ ID NO: 11, CDR2 comprising the amino acid sequence shown in SEQID NO: 12 and CDR3 comprising the amino acid sequence shown in SEQ IDNO: 13, and the heavy chain variable region comprises CDR1 comprisingthe amino acid sequence shown in SEQ ID NO: 14, CDR2 comprising theamino acid sequence shown in SEQ ID NO: 15 and CDR3 comprising the aminoacid sequence shown in SEQ ID NO: 16; or (4) the light chain variableregion comprises CDR1 comprising the amino acid sequence shown in SEQ IDNO: 11, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 12and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13, andthe heavy chain variable region comprises CDR1 comprising the amino acidsequence shown in SEQ ID NO: 14, CDR2 comprising the amino acid sequenceshown in SEQ ID NO: 15 and CDR3 comprising the amino acid sequence shownin SEQ ID NO: 16, except that 1 to 3 amino acids are substituted,deleted, inserted, and/or added in at least one amino acid sequenceselected from the group consisting of the amino acid sequences shown inSEQ ID NOs: 11, 12 and 13, and/or 1 to 3 amino acids are substituted,deleted, inserted, and/or added in at least one amino acid sequenceselected from the group consisting of the amino acid sequences shown inSEQ ID NOs: 14, 15 and 16; (5) the light chain variable region comprisesCDR1 comprising the amino acid sequence shown in SEQ ID NO: 24, CDR2comprising the amino acid sequence shown in SEQ ID NO: 25 and CDR3comprising the amino acid sequence shown in SEQ ID NO: 26, and the heavychain variable region comprises CDR1 comprising the amino acid sequenceshown in SEQ ID NO: 27, CDR2 comprising the amino acid sequence shown inSEQ ID NO: 28 and CDR3 comprising the amino acid sequence shown in SEQID NO: 29; or (6) the light chain variable region comprises CDR1comprising the amino acid sequence shown in SEQ ID NO: 24, CDR2comprising the amino acid sequence shown in SEQ ID NO: 25 and CDR3comprising the amino acid sequence shown in SEQ ID NO: 26, and the heavychain variable region comprises CDR1 comprising the amino acid sequenceshown in SEQ ID NO: 27, CDR2 comprising the amino acid sequence shown inSEQ ID NO: 28 and CDR3 comprising the amino acid sequence shown in SEQID NO: 29, except that 1 to 3 amino acids are substituted, deleted,inserted, and/or added in at least one amino acid sequence selected fromthe group consisting of the amino acid sequences shown in SEQ ID NOs:24, 25 and 26, and/or 1 to 3 amino acids are substituted, deleted,inserted, and/or added in at least one amino acid sequence selected fromthe group consisting of the amino acid sequences shown in SEQ ID NOs:27, 28 and
 29. 24. The method according to claim 23, wherein (1′) thelight chain variable region comprises the amino acid sequence shown inSEQ ID NO: 17, and the heavy chain variable region comprises the aminoacid sequence shown in SEQ ID NO: 18; (3′) the light chain variableregion comprises the amino acid sequence shown in SEQ ID NO: 19, and theheavy chain variable region comprises the amino acid sequence shown inSEQ ID NO: 20; or (5′) the light chain variable region comprises theamino acid sequence shown in SEQ ID NO: 30, and the heavy chain variableregion comprises the amino acid sequence shown in SEQ ID NO: 31.